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MTT Assay Protocol

The document outlines the preparation of MTT solution and solubilization solution for conducting an MTT assay. It includes detailed steps for dissolving MTT, filter sterilization, and storage, as well as the protocol for performing the assay in 96-well plates. The reference provided is a guide on cell viability assays from the Assay Guidance Manual.

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4 views

MTT Assay Protocol

The document outlines the preparation of MTT solution and solubilization solution for conducting an MTT assay. It includes detailed steps for dissolving MTT, filter sterilization, and storage, as well as the protocol for performing the assay in 96-well plates. The reference provided is a guide on cell viability assays from the Assay Guidance Manual.

Uploaded by

adiitu
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Reagent Preparation

MTT Solution
1. Dissolve MTT in Dulbecco’s Phosphate Buffered Saline, pH=7.4 (DPBS) to 5 mg/ml.
2. Filter-sterilize the MTT solution through a 0.2 µM filter into a sterile, light protected
container.
3. Store the MTT solution, protected from light, at 4°C for frequent use or at -20°C for
long term storage.

Solubilization Solution
1. Choose appropriate solvent resistant container and work in a ventilated fume hood.
2. Prepare 40% (vol/vol) dimethylformamide (DMF) in 2% (vol/vol) glacial acetic acid.
3. Add 16% (wt/vol) sodium dodecyl sulfate (SDS) and dissolve.
4. Adjust to pH = 4.7
5. Store at room temperature to avoid precipitation of SDS. If a precipitate forms, warm
to 37°C and mix to solubilize SDS.

MTT Assay Protocol


1. Prepare cells and test compounds in 96-well plates containing a final volume of 100
µl/well.
2. Incubate for desired period of exposure.
3. Add 10 µl MTT Solution per well to achieve a final concentration of 0.45 mg/ml.
4. Incubate 1 to 4 hours at 37°C.
5. Add 100 µl Solubilization solution to each well to dissolve formazan crystals.
6. Mix to ensure complete solubilization.
7. Record absorbance at 570 nm.

Reference:
Riss TL, Moravec RA, Niles AL, et al. Cell Viability Assays. 2013 May 1 [Updated 2016 Jul
1]. In: Markossian S, Grossman A, Arkin M, et al., editors. Assay Guidance Manual [Internet].
Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational
Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK144065/

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